Type 1 diabetes is an autoimmune disease characterized by T lymphocyte-mediated eradication of pancreatic islet beta cells. Studies utilizing the nonobese diabetic (NOD) mouse model of the disease have shown that beta cell-cytotoxic CD8+ T cells are required for diabetes development. Such effectors are also suspected contributors to beta cell elimination in both new-onset and graft-recurrent type 1 diabetes patients. In collaboration with Dr. David Serreze (The Jackson Laboratory), we have developed an approach by which highly pathogenic beta cell-autoreactive CD8 + T cell clones can be derived from early insulitic lesions of NOD mice. Using these clones, we have established that during the early prediabetic period in NOD mice, the pathogenic CD8 + T cell population recognizes at least three distinct beta cell peptides presented by murine class I major histocompatibility complex (MHC) molecules. To more directly link our studies to the disease in humans, we now propose to examine beta cell-autoreactive T cells restricted to the human class I MHC molecule HLA-A2, which appears to be a diabetes susceptibility marker in certain populations. Dr. Serreze has found that diabetes development is accelerated in NOD mice transgenically expressing HLA-A2, and that HLA-A2-restricted beta cell-autoreactive T cells can be isolated from early insulitic lesions of these NOD.HLA-A2.1 mice. We propose to identify synthetic ligands for these T cells using peptide combinatorial libraries in positional scanning format. This will enable us to explore the diversity of antigenic specificities exhibited by HLA-A2-restricted T cells during the initial autoimmune attack on the beta cell. Additionally, for at least some of our T cell clones, the data obtained from screening of the libraries should allow identification of the clone's natural antigen. We will also assess, in both NOD.HLA-A2.1 mice and in HLA-A2-positive type 1 diabetes patients, the disease relevance of the identified ligands. Three Specific Aims are proposed: (1) To identify synthetic peptide ligands for a series of beta cell-autoreactive HLA-A2-restricted T cell clones by screening of positional scanning combinatorial libraries, and to use the data obtained from screening of the libraries in an attempt to identify each clone's natural ligand; (2) To determine the disease relevance of the defined T cell specificities using peptide/HLA-A2 tetramer studies and tolerization regimens in NOD.HLA-A2.1 mice; (3) To use peptide/HLA-A2 tetramer reagents to test the hypothesis that HLA-A2-positive type 1 diabetes patients will exhibit overlapping reactivities to those seen in NOD.HLA-A2.1 mice. Completion of the proposed Aims will result in an improved understanding of the pathogenesis of type 1 diabetes and could conceivably suggest new peptide-based strategies for the preservation of beta cell mass and assays to monitor the autoimmune status of patients.